Despite great effort and resources for the eradication of malaria
this disease still remains a grave public health problem involving
hundreds of thousands of deaths annually (WHO 2010). While research on
vaccines is at an advanced stage, drug therapy is still the principle
tool for the control and eradication of the disease. The emergence of
strains of Plasmodium falciparum and P. vivax which are resistant to
first and second line antimalarials (multidrug resistant or MDR) have
motivated the search for new drugs representing new and distinct
chemical classes and mechanisms of action than those of the antimalarial
drugs currently in use. Chemical compounds of novel structure and of
natural origin represent a major source for the discovery and
development of new drugs for diseases, especially malaria (Kaur et al.
2009; Schmidt et al. 2012a, b).

Historically, plants used in traditional medicine as antimalarials
and febrifuges have provided substances which have proved to be useful
as antimalarials or have served chemists as structural models for the
development of semi-synthetic drugs or purely synthetic analogs. This is
true of the most important antimalarial natural products revealed to
date: quinine (isolated from the bark of Cinchona spp.) and artemisinin
(isolated from Artemisia annua leaves). The therapeutic efficacy and
complex molecular structure of quinine lead to the development of purely
synthetic analogs chloroquine, primaquine, mefloquine, among others in
the last century. More recently, semi-synthetic derivatives (e.g. sodium
artesunate, artemether, arteether and dihydroartemisinin) prepared in
one or more steps from isolated artemisinin have become key
pharmaceutical components in formulations used in what is commonly
called artemisinin combination therapy (ACT) for the treatment of
resistant and MDR Plasmodium falciparum infections (Plowe 2009: Willcox
2011). The extracts of a large number of plant species including many
that are used in traditional medicine have been evaluated for in vitro
antiplasmodial activities and some have also been tested in in vivo
models, usually in mice infected with Plasmodium berghei, P. yoelii or
P. chabaudi. In some cases, the constituent(s) responsible for their
activities have been isolated but relatively few have been studied
further to assess their potential as lead compounds for the development
of new antimalarial drugs (Wright 2005).

In recent years, the monoterpene indole alkaloid ellipticine (1,
Fig. 1) has been the subject of a number of pharmacological studies and
its derivatives have been studied in clinical trials against different
forms of cancer. Ellipticine has been isolated from the alkaline ethanol
extract of the bark of the Amazonian tree Aspidosperma vargasii
(Apocynaceae) (Andrade-Neto et al. 2007; Henrique et al. 2010) which is
used in traditional medicine as an antimalarial (Oliveira et al. 2003).
In vitro antiplasmodial activity of I was first reported by Andrade-Neto
et al. (2007). Recently, the antimalarial activity of 1 was
independently confirmed and the comparable or superior activity of four
derivatives of 1 against P. falciparum in vitro was described (Passemar
et al. 2011; Pohlit et al. 2012).

The roots of the West African climbing shrub Cryptolepis
san-guinolenta (Lindl.) Schltr. (Apocynaceae) are a traditionally used
herbal for malaria treatment. Dry aqueous root extracts of C.
san-guinolenta have proven efficacy according to clinical trials
(Willcox 2011). Cryptolepine (2a, Fig. 1) is the major alkaloid
constituent in the roots of this plant and 2a sulfate exhibits in vitro
activity ([IC.sub.50] = 0.44 [micro]M) against multidrug-resistant K1
strain of P. falciparum (Wright et al. 2001). However, 2a sulfate failed
to cure malaria in mice when given orally and is toxic at a dose of 20
mg/kg to P. berghei-infected mice when administered intraperitoneally
(i.p.) (Cimanga et al. 1997; Wright et al. 2001). Also, 2a has in vivo
chronic effects causing necrosis of rodent liver cells at a dose of 30
mg/kg and also damages the DNA of lymphocytes in vitro. These and other
experimental results do not support the use of the indole alkaloid
cryptolepine (2a) as an antimalarial (Gopalan et al. 2011; Willcox
2011).

Derivatives of cryptolepine (2a) have been introduced having
greater antiplasmodial activity and better toxicity profiles. One of the
most promising of these derivatives is 2,7-dibromocryptolepine which has
potent in vitro activity ([IC.sub.50] = 50 nM) against K1 strain of P.
falciparum and exhibits significant suppression (89-91%) of P. berghei
growth in infected mice at doses of 20-25 mg/kg/day over 4 days. While
2,7-dibromocryptolepine does not apparently intercalate DNA bases as
does 2a, both these compounds damage lymphocyte DNA based on results of
the comet assay (Wright et al. 2001; Gopalan et al. 2011). Recently,
cryptolepine triflate (2a triflate) and ten synthetic analogs of 2a
containing aminoalkyl side chains at C-11 were synthesized and screened
for in vitro antiplasmodial activity and cytotoxicity. One of the most
promising of these compounds was 11-(4-piperidinamino)cryptolepine
hydrogen dichloride (2b) which exhibited potent inhibitory activity
([IC.sub.50] =44 nM) against P. falciparum W2 strain and was the least
toxic of all the compounds tested, including 2a triflate and had the
largest cytotoxicity to antiplasmodial inhibition ratio (46.4:1)
(Lavrado et al. 2008, 2011).

Olivacine (3) is a rare alkaloid which is isolated from
Aspidosperma olivaceum. The antitumor activity of 3 has been the subject
of studies for decades. Compound 3 and analogs have also been
synthesized (Besselievre & Husson 1981; Chevallier-Multon et al.
1990; Guillonneau et al. 2005).

Mechanistic studies demonstrate that cryptolepine (and it analogs)
and ellipticine (1) may have important inhibitory effects on the
formation of hemozoin in P. falciparum. Hemozoin formation is a
fundamental process related to the survival of this parasite within the
red blood cell. Heme is toxic to Plasmodium spp. and is a by-product of
digestion of hemoglobin by proteolytic enzymes in the parasite's
digestive vacuole. Crystallization of heme to hemozoin is a detoxifying
process that occurs naturally within the Plasmodium digestive vacuole
and is necessary for the proliferation of these parasites within the red
blood cell. Inhibition of hemozoin formation is associated with death of
parasites within the red blood cell due to osmotic imbalances and other
effects. Early mechanistic studies involving chemical assays showed that
ellipticine (1) can inhibit heme crystal growth (that is, hemozoin
formation) in the lab (Chong and Sullivan 2003). In other work, it was
presumed that cryptolepine (2a) interacts directly with heme molecules
in the process of inhibiting hemozoin formation (Kumar et al. 2007;
Lavrado et al. 2011).

Previous studies point to the fact that large or small structural
differences among analogous indole alkaloids, such as cryptolepine
analogs and [beta]-carbolines (harmane analogs), can lead to large
differences in in vitro and in vivo antimalarial activity and
cytotoxicity of these compounds. Thus, small structural differences
probably modulate and ultimately define the primary mechanisms of action
of these compounds (DNA intercalation, inhibition of heme
polymerization, inhibition of protein synthesis, among other mechanisms
yet to be revealed) (Arzel et al. 2001; Ancolio et al. 2002; Van Baelen
et al. 2009).

In the present study, the antiplasmodial activity of structurally
related indole alkaloids ellipticine (1), cryptolepine derivative 2b and
olivacine (3) is investigated for the first time in P. berghei-infected
mice and the data are compared to those for cryptolepine triflate (2a
triflate). The in vitro antiplasmodial activity against
chloroquine-resistant and chloroquine sensitive strains ofP. falciparum
and cytotoxicity of these compounds was evaluated in vitro against
murine macrophages as a means to comparatively evaluate selectivity of
the antimalarial effect. The overall aim of this work was to provide
comparative in vitro and especially in vivo antimalarial data for all
four compounds which might lead to insights into the relative importance
of cryptolepine and ellipticine ring systems/skeletons for the further
development of antimalarials.

Materials and methods

Chemicals

Ellipticine (1) used in this work was obtained from two sources.
Synthetic 1 was purchased from Sigma--Aldrich (Steinheim, Germany).
Also, 1 was isolated from the bark of Aspidosperma vargasii from
INPA's Ducke Reserve in Amazonas State, Brazil through an alkaline
extraction sequence followed by column chromatography as described
previously (Andrade-Neto et al. 2007; Henrique et al. 2010).
Cryptolepine triflate (2a triflate) and cryptolepine analog 2b were
obtained by synthesis as described previously (Lavrado et al. 2008).
Olivacine (3) was isolated from Aspidosperma olivaceum from Minas Gerais
State, Brazil, by acid-base extraction. The purity of these compounds
was checked by TLC, UPLC-MS and NMR and was > 98%.

Culture and test for in vitro inhibition of P. falciparum parasites

Strains of P. falciparum used in this study were the antimalarial
drug-susceptible 3D7 clone of the NF54 isolate and the
chloroquine-resistant, pyrimethamine-resistant and cycloguanil-resistant
K1 strain. Parasites were cultured according to the method of Trager and
Jensen (1976) as modified by Andrade-Neto et al. (2007). The parasite
culture was carried out at 37 [degrees]C with a hematocrit of 3-5% and
in an atmosphere of 5% [CO.sub.2]. The parasites were maintained in
vitro in A+ human red blood cells. The culture medium was RPM! 1640
(Sigma-Aldrich) supplemented with 10% human serum and containing 25 mM
HEPES and 2 mM glutamine. The micro-test was performed using the method
of Rieckmann et al. (1978) with modifications which were described in
Andrade-Neto et al. (2007). Stock solutions of indole alkaloids
ellipticine (1), cryptolepine triflate (2a triflate), olivacine (3) and
11-substituted cryptolepine hydrogen dichloride analog 2c were prepared
in DMSO at a concentration of 5.0 mg/mi. Seven dilutions were performed
in culture medium (RPM1 1640) of each stock sample solution resulting in
final test concentrations (well concentrations) of 50-3.2 x [10.sup.-3]
[micro]g/ml. Each diluted sample was tested in duplicate in 96-well test
plates containing a suspension of parasitized red blood cells ata
hematocrit of 3% and initial para-sitemia of 1% of synchronized young
trophozoites (ring forms). The final volume of each well was 200
[micro]l. Reference antimalarial compounds chloroquine and quinine were
used as positive controls at concentrations recommended by WHO (2001).
The test plate was incubated for 48 h at 37 [degrees]C under the same
low oxygen gas mixture used for parasite culture. After the incubation
period, thin smears were prepared from the contents of each well and
evaluated using a microscope. The half maximal inhibitory ([IC.sub.50])
responses compared with the drug-free controls were estimated by
interpolation using Microcal Origin [R] software.

Test for in vivo suppression of Plasmodium berghei

In vivo antimalarial activity was evaluated using P. berghei NK65
strain (drug-sensitive). This strain was maintained by successive
passages of blood forms from mouse to mouse. The test protocol is based
on the 4-day suppressive test as described by Peters (1965). Female
Webster Swiss mice weighing 26 [+ or -] 2 g were used in this study.
Animals were infected intraperitoneally with 0.2 ml of infected blood
suspension containing 1 x [10.sup.5] parasitized erythrocytes and
randomly divided into groups of three individuals. Test groups were
treated orally and subcutaneously at doses ranging from 100 to 1
mg/kg/day. Positive control groups received a dose of 10 mg
chloroquine/kg/day orally or subcutaneously and negative control groups
received 0.2 ml of 2% DMSO or saline. The animals were treated for 4
days starting 24 h after inoculation with P. berghei. On days 5 and 7
after inoculation with parasites, blood smears were prepared from all
mice, fixed with methanol, stained with Giemsa dye, then microscopically
examined (1000x magnification). Parasitemia was determined in coded
blood smears by randomly counting 2000-4000 erythrocytes in the case of
low par-asitemias ([less than or equal to] 10%); or up to 1000
erythrocytes in the case of higher parasitemias. Overall mortality was
monitored daily in all groups during a period of 4 weeks following
inoculation. The difference between the average parasitemia of control
groups (100%) and test groups was calculated as a percentage of parasite
growth suppression (PGS) according to the equation: PGS = 100 x
(A--B)/A, where A is the average parasitemia of the negative control
group and B corresponds to the parasitemia of the test group.

Cytotoxicity test

For this test, macrophages from Swiss mice were used which were
described in Mota et al. (2012), with modifications. The macrophages
were obtained at the time of use by collection with cold, sterile
phosphate saline solution (PSB) from the exudates of the peritoneal
cavity of mice. After centrifuging the peritoneal exudate solutions, the
supernatant was discarded and pellet was resuspended in 5 ml of RPMI
medium without FBS for counting the macrophages in a Neubauer chamber. 1
x [10.sup.5] cells were added to each well. The plate was incubated in a
[CO.sub.2] incubator at 37 [degrees]C for 24 h. The cytotoxicity of the
samples was determined using the methylthiazoletetrazolium (MU)
colorimetric assay (Mosmann 1983). For the assays, the cells were
trypsinized, washed, suspended in DMEM, and distributed into 72 wells
per plate (5 x [10.sup.3] cells per well) then incubated for 18 h at 37
[degrees]C. The samples were separately diluted in DMSO and tested in
triplicate at the following concentrations: 1.5, 3.1, 6.3, 12.5, 25, 50,
and 100 [micro]g/ml. In parallel, we evaluated a control group
consisting of RPMI 1640 without RS, a control group consisting of 1%
DMSO (vehicle) and a positive control (chloroquine, BS Pharma, Belo
Horizonte, MG, Brazil) at the same concentrations used for substances
1-3. After 24 and 48 h of incubation at 37 [degrees]C, 100 [micro]l of
MU (5 mg/m1 in RPMI 1640 without FBS and without phenol red) was added
to each well. After 3 h in a [CO.sub.2] incubator at 37 [degrees]C, the
supernatant was removed and added to 100 [micro]l DMSO in each well. The
absorbance of each well was obtained from a spectrophotometric reading
at 562 nm. The minimum lethal doses that inhibited 50% of cell growth
were obtained from the drug concentration response curves. Results are
expressed in mean [+ or -]standard deviation.

Selectivity index

The relative cytotoxicity to antiplasmoclial activity for a given
compound was evaluated as a selectivity index (SI), where SI =
[IC.sub.50]( murine macrophages)/[IC.sub.50](P.falciparum).

Animals and ethical approval

Adult Webster Swiss albino mice (26 [+ or -] 2g weight) were used
for the antimalarial and toxicity tests and received water and food ad
libitum. In vivo tests were performed using Guidelines for Ethical
Conduct in The Care and Use of Animals of Federal University of Rio
Grande do Norte (CEUA 043/2010).

Results

Indole alkaloids ellipticine (1), cryptolepine triflate (2a
triflate) and olivacine (3) and synthetic analog
1144-piperidinamino)cryptolepine hydrogen dichloride (2b) were assayed
for in vitro activity against Plasmodium falciparum 1<1 e 3D7 strains
and cytotoxic activity against murine macrophages. From the [IC.sub.50]
values for each substance against murine macrophages and malaria
parasite strains it was possible to determine selectivity indices. The
in vitro results are presented in Table 1. Olivacine (3) is a previously
known indole alkaloid for which antimalarial activity has not been
previously described. It was the least active compound in vitro,
however, it did significantly inhibit P. falciparum growth ([IC.sub.50]
= 1.2 [micro]M against K1 strain). The potent in vitro activity of
ellipticine (1) reported previously was confirmed herein ([IC.sub.50]
values of 0.81 and 0.35 [micro]M against P. falciparum K1 and 3D7
strains, respectively). Ellipticine (1) and its structural isomer
olivacine (3) were the least cytotoxic compounds in this study
([IC.sub.50] >0.41 mM, highest concentration tested). This low
cytotoxicity contributed greatly to the high selectivity indices
obtained for 1 and 3 against P. falciparum 3D7 (>1.2 x [10.sup.3] and
>3.4 x [10.sup.2], respectively). The compound with the most in vitro
activity against P. falciparum was rationally designed
11-(4-pipericlinamino)cryptolepine hydrogen dichloride (2b) ([IC.sub.50]
=0.10 and 0.087 [micro]M, against P. falciparum 1<1 and 3D7 strains,
respectively) which had been selected from among a number of synthetic
cryptolepine analogs reported earlier based on its favorable in vitro
antimalarial and cytotoxic properties reported in that earlier work
(Lavrado et al. 2008). In the present work, cryptolepine analog 2b
exhibited an [IC.sub.50] value against murine macrophages of 34[micro]M
thus making it the second most cytotoxic compound after 2a triflate.
Relatively high cytotoxicity against murine macrophages lead to 2a
triflate and 2b exhibiting the lowest SI values.

The indole alkaloids 1-3 were evaluated in vivo in P.
berghei-infected mice in the 4-day suppressive test and the result is
presented in Table 2. Ellipticine (1) was highly active at an oral dose
of 50 mg/kg/day (100% inhibition versus controls on days 5 and 7). At
this same dose, the mean survival time (MST) of the animals was >40
days (limit of the observation period and identical to the MST of the
control substance chloroquine). Also, 1 had good oral activity on day 5
and good activity via subcutaneous injection on day 7 at 10 mg/kg/clay
(77 and 70% inhibition, respectively; MST= 27-29 days) and moderate oral
activity at 1 mg/kg/day (61-67% inhibition, MST= 22-23 days). 3
exhibited high oral activity at [greater than or equal to]50 mg/kg/day
(90-97% inhibition, MST= 23-27 days) and low to moderate oral and
subcutaneous activity at 1 and 10 mg/kg/day (7-64% inhibition, MST=
24-27 days). Cryptolepine triflate (2a triflate) exhibited only moderate
oral and subcutaneous activity at 50 mg/kg/day (43-63% inhibition, MST=
24-25 clays). At a dose of 50 mg/kg/day, subcutaneously injected
cryptolepine derivative 2b was lethal to infected mice (MST= 3 days) and
oral activity at this dose was moderate (45-55% inhibition, MST= 25
days). At 10 mg/kg/day, 2b administered orally and subcutaneously
exhibited low to moderate activity (25-60% inhibition, MST= 24 days).

The in vitro activity of 1 against P. folciparurn was described for
the first time by Anclrade-Neto (2007) ([IC.sub.50] = 73 nM, K1 strain)
and was recently independently confirmed against the
chloroquine-resistant FcM29-Cameroon strain of P. falciparurn
([IC.sub.50] = 1.13 pLM ) (Passemar et al. 2011; see also Pohlit et al.
2012). Presented herein are the first data on the antimalarial activity
of olivacine (3) which exhibited important in vitro antimalarial
activity and low cytotox-icitv.

Lavrado et al. (2008) synthesized analogs of 2a containing
diamino-alkane side chains at C-11. The basis for this approach was the
observation that a basic amino side chain is a requirement for
chloroquine accumulation in the acidic digestive vacuole of the
parasite. These analogs of 2a were potent inhibitors ([IC.sub.50] =
20-455 nM) of P. falciparum strains having different drug resistance
phenotypes (Lavrado et al. 2008, 2011). Herein, the rationally designed
11-(4-piperidinamino)cryptolepine 2b which has optimal in vitro
antimalarial and SI (Lavrado et al. 2008) was the most active compound
against P. falciparum in vitro.

Ellipticine (1), olivacine (3) and related ellipticine-like
compounds have received attention due to their high toxicity to tumor
cells and low number of side effects. Thus, derivatives of these
compounds are excellent target compounds for clinical studies (Sizum et
al. 1988; Jasztold-Howorko et al. 2004). In tumor cells, there is
evidence that the mechanism of action involves DNA intercalation and
interference with the activity of topoisomerase II with consequent
cytotoxic effects which are related to size, shape and flatness of 1 and
3 (Carvalho and Laks 2001; Braga et al. 2004). Importantly, we observed
low cytotoxicity for 1 and 3 against mouse macrophages whereas 2a
triflate and cryptolepine derivative 2b exhibited relatively high
toxicity to macrophages.

As seen above, compounds 1-3 inhibit P. falciparum in vitro. Only
cryptolepine has been studied previously using in vivo antimalarial
models. So, the effects of 1-3 in P. berghei-infected mice were
explored. Ellipticine (1) was the most active compound in vivo
suppressing parasitemia by 100% and providing MST of >40 days at an
oral dose of 50 mg/kg/day. Remarkably, this was the same result obtained
for control compound chloroquine at 10 mg/kg/day for all animals. Also,
up to 77% inhibition of parasitemia was observed for 1 at doses of 10
mg/kg/day. Few compounds (e.g. chloroquine) significantly reduce
parasitemia in mice infected with P. berghei. At the highest dose of 1
(50 mg/kg/day), no mortality or other signs of intoxication were
observed. The in vivo antimalarial activity of 3 was lower than that of
its structural isomer 1. No toxic effects were observed in mice which
were administered 3 at up to 100 mg/kg/day.

Cryptolepine analog 2b exhibited moderate activity in vivo and
acute toxicity. Orally at 50 mg/kg/day, 2b inhibited the growth of P.
berghei by 55%. However, subcutaneously at 50 mg/kg/day, 2b killed all
mice after the second dose. Subcutaneously, at 10 mg/kg/day, 2b caused
ulceration at the place of injection and inhibited P. berghei only
moderately (60%).

Cryptolepine triflate (2a triflate) and derivatives (e.g.
2,7-dibromocryptolepine and 2b) exhibit limited efficacy in P.
berghei-infected mice. Also, cytotoxicity, acute toxicity and
genotoxicity are potential drawbacks to their use as drugs. Importantly,
structural isomers 1 and 3 exhibited good in vitro SI and 1 provided P.
berghei-infected mice with mean survival times greater than 40 days.
These data reveal the potential of ellipticine (1) and olivacine (3) as
antimalarial leads.